Targeted mutagenesis in Spirulina
Inventors
Takeuchi, Ryo • Roberts, James
Assignees
Publication Number
US-10415013-B2
Publication Date
2019-09-17
Expiration Date
2035-09-09
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Abstract
This disclosure describes techniques for creating stable, targeted mutations in Spirulina (Athrospiria) and Spirulina having stable, targeted mutations.
Core Innovation
This invention describes methods for introducing stable, targeted mutations into the genome of Spirulina, a genus of cyanobacteria. The disclosed techniques involve contacting Spirulina cells with an osmotic stabilizer, exposing them to a vector that includes homology arms for site-specific homologous recombination, and inducing artificial competence in the Spirulina to facilitate uptake and integration of genetic material. The invention also includes Spirulina strains that carry such stable, targeted mutations, which can persist through multiple generations.
The need addressed by this invention stems from the historical difficulty of achieving stable and targeted genetic modifications in Spirulina, which is neither naturally competent for DNA uptake nor receptive to standard bacterial transformation methods. Traditional methods such as electroporation and random mutagenesis have either failed to provide stable integration of foreign DNA at predetermined sites in the Spirulina genome, or resulted only in random mutations. The invention solves this problem by combining osmotic stabilization, artificial competence induction, and the use of vectors with homology arms to achieve precise genetic changes that are stably inherited.
The methods allow for a variety of targeted genomic modifications in Spirulina, such as gene deletions, additions (including exogenous or endogenous genes, and regulatory elements), and the insertion of sequences that confer desirable traits. Additionally, the invention outlines specific embodiments for modifying Spirulina to overproduce commercially valuable compounds, such as pigments or lipids, by precisely altering its genome using the described transformation and recombination techniques.
Claims Coverage
The independent claim focuses on a method for preparing a composition containing an extract of Arthrospira, particularly defined by a specific ratio of C-phycocyanin to allophycocyanin, with key inventive features relating to steps in biomass preparation and extraction.
Preparation of Arthrospira extract with high C-phycocyanin to allophycocyanin ratio
The invention claims a method for preparing a composition comprising an extract of Arthrospira, where the composition has a ratio of C-phycocyanin to allophycocyanin of at least 5:1 on a weight basis. The method steps include: 1. Partially drying Arthrospira biomass to a slurry containing not less than 50% water by weight. 2. Lysing the suspension. 3. Removing the solid components from the suspension to form a crude extract. Each step and the specific ratio are essential features of the method, providing a composition enriched in C-phycocyanin relative to allophycocyanin.
The claim coverage centers on a method for producing a Spirulina (Arthrospira) extract with a significantly elevated ratio of C-phycocyanin to allophycocyanin, defined by the particular processing steps and compositional parameters.
Stated Advantages
The technique enables creation of stable, targeted mutations in Spirulina, which were previously unachievable using existing transformation methods.
The methods allow for efficient generation of Spirulina strains with desired genetic modifications that are heritable for at least ten generations.
The invention permits the targeted addition, deletion, or disruption of genes or regulatory elements in Spirulina, enabling tailored production of compounds of interest.
Use of the disclosed methods improves the ability to stably integrate exogenous DNA at pre-determined chromosomal locations in Spirulina.
Documented Applications
Production of Spirulina strains with increased synthesis of neutral lipids (such as triglycerides and wax esters) for biofuel and specialty chemical feedstocks.
Creation of Spirulina strains with reduced glycogen accumulation for redirecting carbon flow to other biosynthetic pathways, including lipid and fatty acid synthesis.
Modification of carotenoid biosynthesis in Spirulina for increased or altered production of pigments such as zeaxanthin, astaxanthin, and canthaxanthin.
Engineering Spirulina strains to overproduce phycocyanin or phycoerythrin for use as cosmetic, food, or medical imaging pigments.
Preparation of extracts from Arthrospira (Spirulina) biomass with a high ratio of C-phycocyanin to allophycocyanin for use in various compositions.
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